Diketoacid-derivatives as inhibitors of polymerases

ABSTRACT

Diketoacids of Formula (A) are useful as inhibitors of viral polymerases. In particular hepatitis C virus RNA dependent RNA polymerase (HCV RdRp), hepatitis B virus polymerase (HBV pol) and reverse transcriptase of human immunodeficiency virus (HIV RN.) The group R may be broadly chosen and is an organic moiety which contains 2 to 24 carbon atoms and includes an optionally cyclic or heterocyclic group in which the atom directly bonded to the adjacent carbonyl in the diketoacid is part of the ring structure.

The present application claims priority of U.S. provisional applicationSer. No. 60/096,528, filed Aug. 13, 1998. The present application is a371 of PCT/GB99/02446, filed Jul. 27, 2999.

TECHNICAL FIELD

The present invention relates to compounds useful as enzyme inhibitors,in particular as inhibitors of enzymes involved in the transfer ofphosphoryl groups and, especially as inhibitors of polymerases. Theinvention further relates to pharmaceutical compositions containing suchcompounds, and to their use in the treatment of viral infections.

Polymerases are the enzymes which catalyse the formation ofphosphodiester bonds in RNA and DNA. They play an essential role inviral replication and, therefore, are an important target in the fightagainst viral diseases such as human immunodeficiency virus (HIV),hepatitis, and poliomyelitis.

BACKGROUND ART

U.S. Pat. No. 5,475,109 describes dioxobutanoic acids substituted withpiperidine or similar N-substituted saturated cycloalkyls as inhibitorsof the cap-dependent endonuclease of influenza virus.

DISCLOSURE OF THE INVENTION

The present inventors have discovered that a range of diketoacids haveutility as enzyme inhibitors and, in particular, as polymeraseinhibitors and more particularly as inhibitors of hepatitis C NS5RNA-dependent RNA polymerase, HBV DNA-dependent RNA polymerase and HIVDNA-dependent DNA polymerase. Their investigations indicate that thesecompounds may act by interfering with the binding of phosporyl groups atthe active site of the enzyme and may, therefore, have broad applicationin inhibiting enzymes involved in the transfer of phosphoryl groups.

According to a first aspect of the present invention there is provided acompound of formula A shown below. This compound is suitable fortherapeutic use, for instance as an enzyme inhibitor.

Optionally, the compound may be in the form of a pharmaceuticallyacceptable salt or ester, which can be hydrolysed in vivo to thecorresponding diketoacid.

In formula A, the group R is an organic moiety which contains from 2 to24, preferably 4 to 20, most preferably 6 to 17 carbon atoms in total. Rincludes an optionally substituted cyclic or heterocyclic group in whichthe atom directly bonded to the adjacent carbonyl in the diketoacid ispart of the ring structure. Preferably, this atom is a carbon atom.

The ring which is thus bonded to the carbonyl group is preferably a 3 to8 membered ring, particularly a 4 to 6 membered ring.

Thus, for example, R may be selected from:

(i) optionally substituted aromatic groups, especially those includingsix membered rings, such as phenyl and naphthyl;

(ii) optionally substituted heteroaryl groups especially those includingfive and six membered rings such as thiophene, pyrrole, furan,imidazole, pyridyl, pyrimidyl, and pyridazyl; the heteroaryl ring may,optionally be fused to another ring;

(iii) optionally substituted cycloalkyl groups, especially thoseincluding five or six membered rings such as cyclopentyl, cyclohexyl andadamantyl;

(iv) optionally substituted cycloalkenyl groups, especially thoseincluding five or six numbered rings such as cyclohexenyl,cyclopentenyl;

(v) optionally substituted cyclic heteroalkyl groups, especially thoseincluding five or six numbered rings such as piperidyl, pyrrolidyl,tetrahydrofuranyl, and tetrahydropyranyl; in this class 4-piperidylrings substituted with an aryl group at carbon 4 and on acyl or sulfonylsubstituent at N1 are preferred.

In the case of optional substitution, one or more substituents may bepresent and a wide variety of substituents are possible. Preferredoptional substituents for all compounds of the present invention are setout in the following list:

(a)—OH;

(b)—SH;

(c)—halogen, such as fluorine, chlorine or bromine,

(d)—CO₂H;

(e)—CN;

(f)—NO₂;

(g)—NR₁R₂ wherein each of R₁ and R₂ is selected from H and lower alkylgroups having 1 to 6 carbon atoms; or R₁ and R₂ together form a ringincluding 4 to 6 carbon atoms;

(h)—SO₂NR₁R₂ where R₁ and R₂ are as defined above;

(i)—CONH₂, —NHCO₂H, or —NHCOCOOH;

(j) an alkyl (or alkenyl or alkynyl group) group having 1 to 12 (2 to12) carbon atoms, preferably 1 to 7 (2 to 7) carbon atoms optionallysubstituted by any one or more of the groups (a)-(i) above and/oroptionally interrupted by a group selected from —O—, —S—, —NR₃—,

—CO₂—, —OCO—, —CONR₃—, —NR₃CONR₃—, —SO₂—, —NR₃SO₂—, and —SO₂NR₃—; whereeach R₃ independently is H or lower alkyl of 1 to 6 carbon atoms;

(k) an aryl or heteroaryl group having 2 to 10 carbon atoms optionallysubstituted with any one or more of groups (a) to (j) above;

(l) an aralkyl or heteroaralkyl group having 3 to 16 carbon atomsoptionally substituted with any one or more of groups (a)-(j) aboveand/or in which the alkyl part of the group is optionally interrupted bya group selected from —O—, —S—, —NR₃—,

—CO₂—, —OCO—, —CONR₃—, —NR₃CONR₃—, —SO₂—, —NR₃SO₂—, and —SO₂NR₃—; whereR₃ is as defined above;

(m)

where R₄ is an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, orheteroaralkyl group as such groups are defined above at (j), (k) and(l);

(n)

where R₄ is as defined above;

(o) —OR₄ where R₄ is as defined above;

(p)

where R₄ is as defined above;

(q) —SO₂R₄ where R₄ is as defined above;

(r) —NHR₄ or —N(R₄)₂ where R₄ is as defined above;

(s) —NHSO₂R₄ or —SO₂NHR₄, where R₄ is as defined above;

(t) —SR₄

and each of optional substituents (j) to (t) above may optionally itselfbe substituted by one or more groups selected from (j) to (t).

A preferred class of compounds of formula A is represented by formula E:

in which Ar is an optionally substituted aryl or heteroaryl group.Optional substituents may be selected from the list of preferredsubstituents set out above. Within this class of preferred compounds twoespecially preferred groups are set out below (formulas F and G)

R₅, R₆, R₇ and R₈ are, independently H or are selected from the optionalsubstituents listed above and R₇ and R₈ taken together may form a 4 to7, preferably 5 or 6 membered ring; and X is O, S, NH, or NR₄ where R₄is as defined above.

In compounds of formula F, (which are optionally substituted phenyldiketoacids) ortho, meta and para substitution are possible.

In general, it is preferred that there is a single substituent,preferably at the position which is ortho- or meta- to the diketoacidgroup. Substitution at the meta-position is especially preferred. Wheretwo substituents are present, then preferably the phenyldiketoacid is2,5-substituted; 3,5-substitution is also possible, as is2,4-substitution provided, in the latter case, that the substituent atthe 4-position is relatively small (e.g. methyl). Disubstitution at the2,3- and 2,6-positions is, in general, not preferred.

Preferred substituents, especially at the ortho and meta positions, areether groups of formula (o) above (i.e. —OR₄), hydroxyl, and —NHSO₂R₄.It is generally preferred that no more than one substituent be —OR₄and/or —NHSO₂R₄.

Preferred examples of —OR₄ groups which may be found at the ortho andmeta positions and particularly at the meta position include:

—OCH₂Ar or, less preferably —O(CH₂)₂Ar where Ar is an optionallysubstituted aryl or heteroaryl group and is particularly preferably anoptionally substituted phenyl group. Examples of preferred substituentson the aryl group, and especially on the phenyl ring include halogens,especially fluorine and chlorine, and electron-withdrawing groups suchas —CN, —CO₂H, and —CF₃ as well as ether and aryl groups;

—O—(CH₂)₃—CN; and

—O—(CH₂)₃—C≡CH.

Preferred sulfonamide groups which may be found at the ortho- and meta-positions, particularly at the meta-position are those of formula:

—NH—SO₂—Ar, where Ar is an optionally substituted aryl or heteroarylgroup, preferably an optionally substituted phenyl group. Preferredoptional substituents for the aryl, preferably phenyl group, include:—CN; halogens, especially chlorine and fluorine, —CF₃, lower (C₁₋₆)alkyl (especially methyl), hydroxy-, ether, and —NO₂ groups.

For both the —OCH₂Ar and —NHSO₂Ar substituted compounds, anotherpreferred example of Ar is naphthyl.

Other preferred substituents at the ortho and meta positions are lower(eg C₁₋₆) alkyl groups, especially C₁₋₄ alkyl, such as methyl and ethyl,but in particular methyl, aralkyl groups, especially phenylmethylgroups, optionally substituted in the phenyl ring, especially by ahalogen, and nitrogen-containing substituents such as primary, secondaryor tertiary amine groups, optionally in protonated form, amide,urethane, or urea groups in each of which examples there is a nitrogenatom bonded to the phenyl ring.

One particularly preferred sub class of compounds of formula F is thosein which each of R₅ and R₆ is selected from H, HO—, R₄O—, and —NHSO₂R₄provided that no more than one of R₅ and R₆ is R₄O— or —NHSO₂R₄.

In compounds of formula G the diketoacid group may be at the 2- or 3-position of the ring. In many cases substitution at the 2-position ispreferred.

Preferred examples of compounds of formula G are those in which the fivemembered aromatic ring,

is a pyrrole or thiophene ring. In the case of the pyrrole-substituteddiketoacids, the groups R₇ and R₈ may both be hydrogen and in many casesthat is preferred. if R₇ and R₈ correspond to substituent groups, thenthese may be at any of the positions not already occupied by thediketoacid group. Examples of possible substituents include alkyl(especially methyl), halogen, and aralkyl (especially benzyl) groups.

One embodiment of pyrrole substituted diketoacid is that in which thediketoacid group is at the 2-position of the ring and where the onlyother substituent in the ring is on the nitrogen atom. In this case,preferred examples of the substituent R₄ present on the nitrogen atom,include alkyl, aryl or aralkyl groups, particularly aralkyl (such asbenzyl) groups. Where an aryl or aralkyl group is present these arepreferably substituted with halogen atoms, such as fluorine or chlorine,or by cyano-groups.

In the case of the thiophene-substituted diketoacids a wide range ofsubstituents R₇ and R₈ may be employed in various positions as will beevident from the tables infra. Preferred thiophenes have an aralkyl(such as optionally substituted benzyl) or aryl (such as optionallysubstituted phenyl) substituent, e.g. at the 5-position of the thiophenering.

Compounds containing furanyl rings may also be useful, especially forinhibiting HIV reverse transcriptase.

Preferred substituents are optionally substituted aryl groups(especially optionally substituted phenyl). Substitution is preferablyat the 5-position of the ring.

The formulae of numerous preferred specific compounds of the presentinvention are presented later below.

The compounds of the present invention having formula A may be preparedby a process which comprises reaction of a compound of formula B with adialkyloxalate of formula C followed by hydrolysis of the resultingdiketo-ester of formula D:

where R′ is an alkyl group, typically having 1-6 carbon atoms. In thecase where the target molecule is a pharmaceutically acceptable ester ofthe compound of formula A then R′ in formula C may be selectedaccordingly, and the step of hydrolysing the compound of formula Domitted, since in vivo hydrolysis can render the compounds active.

Preferred enzymes for inhibition by the compounds of the invention arethose involved in phosphate transfer, in particular polymerases such asDNA polymerases, and RNA polymerases both of which may be either RNAdependent or DNA dependant. Compounds of the invention may particularlypreferably be employed in the inhibition of viral enzymes. Examples ofviral enzymes include RNA-dependent RNA polymerase and reversetranscriptases.

The compounds of the invention may be used as inhibitors of plant oranimal (including human) viruses.

The viruses may be RNA viruses, which may, for example, be positivesingle stranded viruses of which polio virus, hepatitis C virus andencephalomyocarditis are examples, negative single stranded viruses suchas orthomyxoviruses, and paramyxoviruses, and retroviruses of which HIVis a prominent example. Alternatively, the viruses may be DNA viruses,especially double stranded DNA viruses such as hepatitis B virus. Inparticular, compounds of the present invention may inhibit one or moreof the following enzymes: hepatitis C virus RNA dependent RNA polymerase(HCV RdRp), hepatitis B virus polymerase (HBV pol) and reversetranscriptase of human immunodeficiency virus (HIV RT).

Especially preferred compounds of the invention will be suitable for useas HCV RdRp inhibitors.

Other classes of enzyme involved in phosphate transfer which may besusceptible to inhibition by compounds of the present invention includephosphatases, Rnases, integrases and ribozymes.

According to a further aspect of the invention there is provided thenon-therapeutic use of compound of formula A or suitable salt or esteras an enzyme inhibitor, especially as an inhibitor of polymerases,especially viral polymerases. For instance, compounds of the inventionmay be of utility in agriculture and horticulture for treating plantsinfected with or susceptible to plant virus.

According to a further aspect of the invention there is provided the useof a compound of formula A or of a pharmaceutically acceptable salt orester thereof in the manufacture of a medicament for treatment of aviral illness in a human or animal. For instance, the medicament may beused to treat viral illness by inhibiting one or more viral polymerase.Preferably the medicament is for treatment of hepatitis, such ashepatitis B or C, particularly hepatitis C, and human immunodeficiencyvirus.

A still further aspect of the invention provides a pharmaceuticalcomposition comprising a compound of formula A, or a pharmaceuticallyacceptable salt or ester thereof and a pharmaceutically acceptableexcipient, diluent or carrier. The composition may be in any suitableform, depending on the intended method of administration. It may forexample be in the form of a tablet, capsule or liquid for oraladministration, or of a solution or suspension for administrationparenterally.

The pharmaceutical compositions optionally also include one or moreother agents for the treatment of viral infections such as an antiviralagent, or an immunomodulatory agent such as α-, β-, or γ-interferon.

A still further aspect of the invention provides a method of inhibitingan enzyme, especially a viral polymerase and/or of treating orpreventing a viral illness, the method involving administering to ahuman or animal (preferably mammalian) subject suffering from thecondition a therapeutically or prophylactically effective amount of thepharmaceutical composition described above or of a compound of formula Aor salt or ester thereof. “Effective amount” means an amount sufficientto cause a benefit to the subject or at least to cause a change in thesubject's condition.

The dosage rate at which the compound, salt or ester is administeredwill depend on the nature of the subject, the nature and severity of thecondition, the administration method,used, etc. Appropriate values areselectable by routine testing. The compound, salt or ester may beadministered alone or in combination with other treatments, eithersimultaneously or sequentially. For instance, it may be administered incombination with effective amounts of antiviral agents,immunomodulators, anti-infectives, or vaccines known to those ofordinary skill in the art. It may be administered by any suitable route,including orally, intravenously, cutaneously, subcutaneously, etc. Itmay be administered directly to a suitable site or in a manner in whichit targets a particular site, such as a certain type of cell. Suitabletargeting methods are already known.

A further aspect of the invention provides a method of preparation of apharmaceutical composition, involving admixing one or more compound offormula A or salt or ester thereof with one or more pharmaceuticallyacceptable adjuvants, diluents or carriers and/or with one or more othertherapeutically or prophylactically active agents.

Modes for Carrying Out the Invention

Embodiments of the invention are described below by the way of exampleonly.

EXAMPLES

(1) Synthesis

The synthesis of the 2,4-dioxobutanoic acids consists of a Claisencondensation reaction between a methyl ketone substrate and diethyloxalate in the presence of sodium ethoxide in tetrahydrofuran (Scheme1A) and the subsequent hydrolysis of the ethyl ester with sodiumhydroxide in methanol (Scheme 1B)

Reagents: (i) diethyl oxalate/NaOEt in THF

Reagents: (i) 5 eg. NaOH/MeOH

Exemplary Procedure for the Synthesis of the 2,4-dioxobutanoate EthylEsters

(Scheme 1A)

In a 50 ml round bottom flask with a stirring bar and under an inertatmosphere, the methyl ketone compound (1.0 mmole) in 10 ml of drytetrahydrofuran (THF) is reacted with 2 equivalents of diethyl oxalateand 2 equivalents of sodium ethoxide (NaOEt) at ambient temperature for3 hours. When reaction is completed, the reaction mixture is poured intoa 1N aqueous hydrochloric acid (HCl) and extracted with ethyl acetate(EtOAc). The organic phase is separated, washed first with water andthen with brine. The organic layer is dried over sodium sulfate(Na2SO4), filtered and solvent is removed in vacuo leaving the desireddioxobutanoate ethyl ester in quantitative yield.

Exemplary Procedure for Hydrolysis of the Ethyl Ester

(Scheme 1B)

In a 50 ml round bottom flask with a stirring bar, the2,4-dioxobutanoate ethyl ester compound (1.0 mmole) in 10 ml of methanol(MeOH) is reacted with 5 equivalents of sodium hydroxide (NaOH) atambient temperature for 2 hours.

The methanol is removed in vacuo. The aqueous residue is washed withdiethyl ether (Et2O). The aqueous fraction is acidified by addition of1N aqueous hydrochloric acid solution (HCl) and the milky mixture isextracted with two portions of ethyl acetate (EtOAc). The combinedorganic fractions are washed with brine. The organic layer is dried oversodium sulfate (Na2SO4), filtered and solvent is removed in vacuoleaving the desired dioxobutanoic acid product.

Using this or analogous methods, compounds were produced as set out inthe following Tables, which are categorised according to their “R”group.

The Tables include IC₅₀ data and the methods for assay are explainedafter the Tables.

Notes to Table:

NA=not active as an inhibitor at concentrations up to that stated.

ND=not done.

In the tables, where nitrogen atoms appear to be divalent, the presenceof a hydrogen atom is implied.

TABLE I HCV-polymerase inhibitors: examples of 2,5-substitutedphenyldiketoacids

Ex. No. R1 R2 IC 50 (μM) 1

5.6 2

3 3

27.9 4

8 5

17 6

18 7

2.92 8

44 9

51 10

20 11

7.08 12

16.7 13

2.6 14

26 15

83.5 16

4.3 17

11.6 18

2.2 19

11.9 20

0.38 21

0.955 22

19 23

0.94 24

19 25

28 26

26 27

2.84 28

6.2 29

3.9 30

15 31

18 32

6.1 33

18.2 34

9.6 35

6.1 36

1.6 37

18 38

16 39

22 40

8.3 41

28.9 42

16.6 43

20 44

18.5 45

12.9 46

30.1 47

20.7 48

22 49

32 50

7.8 51

1.9 52

10 53

0.115 54

2.3 55

10.8 56

23.6 57

2.1 58

13.6 59

25.3 60

40 61

31 62

10 63

1.7 64

0.23 65

45 66

11 67

16 68

30 69

14 70

9.2 71

10.6 72

0.48 73

5.6 74

3.6 75

19.2 76

50 77

4.8 78

0.67 79

6 80

3 81

1.4 82

19 83

9.4 84

0.95 85

13 86

2.05 87

2.3 88

0.7 89

3.3 90

1.8 91

6.2 92

1 93

1.9 94

5.8 95

0.48 96

50 97

2.8 98

1 99

0.6 100

7.8 101

7 102

1.5 103

6 104

50 105

13.7 106

6.8 107

0.14 108

6.9 109

0.17 110

30 111

0.12 112

1.33 113

0.1 114

0.5 115

3.7 116

0.3 117

0.14 118

0.2 119

0.049 120

0.36 121

4 122

2 123

0.29 124

28 125

0.17 126

0.056 127

0.3 128

24 129

1.6 130

0.14 131

0.78 132

0.67 133

3.2 134

23 135

21 136

0.2 137

0.9 138

1.1 139

1.4 140

1 141

0.56 142

0.4 143

0.45 144

14 145

1.2 146

15 147

1.3 148

0.26 149

0.55 150

2.3 151

0.5 152

20 153

19 154

30

TABLE II HCV-polymerase inhibitors: examples of 3,5- substitutedphenyldiketoacids

IC Ex. 50 No. R1 R2 (μM) 155

1.4 156

1.3 157

0.9 158

0.2 159

20 160

0.1

TABLE III HCV-polymerase inhibitors: examples of 2,4-substitutedphenyldiketoacids

Ex. No. R1 R2 IC 50 (μM) 161

2.8 162

5.5 163

26 164

47 165

2 166

20 167

0.6

TABLE IV HCV-polymerase inhibitors: examples of 2,3- substitutedphenyldiketoacids

Ex. No. R1 R2 IC 50 (μM) 168

18 169

>50 170

>50

TABLE V HCV-polymerase inhibitors: examples of 2,6- substitutedphenyldiketoacids

Ex. No. R1 R2 IC 50 (μM) 171

12 172

>50

TABLE VIa HCV-polymerase inhibitors: examples of pyrrole-2- substituteddiketoacids

Ex. No. R1 IC 50 (μM) 173

21 174

13.4 175

25 176

29 177

25 178

17.9 179

12.8 180

93 181

30 182

30 183

32 184

6.7 185

6.3 186

24 187

36 188

12.7 189

28 190

18

TABLE VIb HCV-polymerase inhibitors: examples of thiophene-2-substituted diketoacids

Ex. No. R1 IC 50 (μM) 191

10 192

8.2 193

12 194

16 195

11.1 196

15 197

11 198

7.9 199

17 200

8.2 201

20 202

68 203

19.8 204

11 205

74 206

65 207

9.9 208

11.6 209

12.6 210

27 211

82 212

7.5 213

5.9 214

17 215

15.3

TABLE VIc HCV-polymerase inhibitors: examples of furan-2- substituteddiketoacids

Ex. R1 IC 50 (μM) 216

50 217

58 218

41.2

TABLE VIIa HCV-polymerase inhibitors: examples of pyrrole- 3-substituteddiketoacids

Ex. No. R1 IC 50 (μM) 219

23.7 220

4.6 221

20.6

TABLE VIIb HCV-polymerase inhibitors: examples ofthiophene-3-substituted diketoacids

Ex. No. R1 IC 50 (μM) 222

4 223

27 224

50 225

167 226

17 227

15 228

17.8 229

80 230

8.6 231

9.4 232

11.8 233

9.2 234

14.5 235

7.5 236

26

TABLE VIIc HCV-polymerase inhibitors: examples of furan-3- substituteddiketoacids

Ex. No. R1 IC 50 (μM) 237

14 238

47.5

TABLE VIII HCV-polymerase inhibitors: examples of alkyl-diketoacids

Ex. No. R1 IC 50 (μM) 239

9.4 240

18 241

37 242

12.8 243

6.7 244

77 245

81.4 246

18 247

45 248

10 249

60 250

17 251

21 252

61 253

55 254

14 255

16.7 256

25 257

50

TABLE IXa most active HCV-inhibitors

Ex. No. R1 HCV HIV HBV 126

0.056 100 ND 160

0.1 NA ND 113

0.1 90 ND 53

0.115 37 ND 111

0.12 80 ND 107

0.14 58 ND 117

0.14 100 ND 109

0.17 NA ND 158

0.2 NA ND 64

0.23 NA ND 116

0.3 NA ND 120

0.36 80 ND 20

0.38 27 ND 72

0.48 NA ND 99

0.6 50 ND 78

0.67 35 ND 88

0.7 NA ND 84

0.95 NA ND 21

1 >50 ND 23

1 59 ND 112

1.33 90 ND 155

1.4 130 416 36

1.6 24 ND 90

1.8 NA ND 165

2 NA ND 18

2.2 30 ND 161

2.8 320 108 80

3 NA ND 27

3 >50 ND 7

3.3 61 6 16

4.3 >100 ND 162

5.5 NA ND 1

5.6 90 NA 103

6 NA ND 243

6.7 26.8 ND 198

7.9 NA ND 4

8 >100 ND 192

8.2 NA ND 66

11 NA ND 19

12 77 ND 179

12.8 NA NA 190

18 NA NA 24

19 71 ND 49

32 NA ND

TABLE IXb most active HBV-Pol-inhibitors

Ex. No. R1 HCV HIV HBV 206

65 NA 2 205

74 NA 3.3 225

167  86 4 202

70 >100 9 196

15 50 9

TABLE IXc most active HIV-RT-inhibitors

Ex. No. R1 HCV HIV HBV 258

>100 3.6 NA 218

41.2 11.8 40 259

>100 16 NA 40

8.3 12 NA 20

0.38 27 ND 8

44 19 ND

2. Measurement of Inhibitory Activity

The effectiveness of the compounds set out above as polymeraseinhibitors, stated above as IC₅₀ values, was assessed in screeningassays as follows.

In initial tests, the compounds were tested to see if they wereeffective as inhibitors of the RNA-dependent RNA polymerase (RdRp) ofhepatitis C virus (HCV). The HCV NS5B protein is the viral RdRp;compounds capable of interfering with the activity of this enzyme arethus expected to block viral replication.

Test for Inhibition of Hepatatis C Virus RdRp

WO96/37619 describes the production of recombinant HCV RdRp from insectcells infected with recombinant baculovirus encoding the enzyme. Thepurified enzyme was shown to possess in vitro RNA polymerase activityusing RNA as template. The reference describes a polymerisation assayusing poly (A) as a template and oligo(U) as a primer. Incorporation oftritiated UTP is quantified by measuring acid-insoluble radioactivity.The present inventors have employed this assay to screen the variouscompounds described above as inhibitors of HCV RdRp and other virallyencoded polymerases.

Incorporation of radioactive UMP was measured as follows. The standardreaction (100 μl) was carried out in a buffer containing 20 mM tris/HClpH 7.5, 5 mM MgCl₂, 1 mM DTT, 50 mM NaCl, 1 mM EDTA, 2 OU Rnasin(Promega), 0.05% Triton X-100, 1 μCi[³H] UTP (40 Ci/mmol, NEN), 10 μMUTP and 10 μg/ml poly(A). oligo (U)₁₂ (1 μg/ml, Genset) was added as aprimer. The final NSSB enzyme concentration was 20 nM. After 1 hourincubation at 22° C. the reaction was stopped by adding 100 μl of 20%TCA and applying samples to DE81 filters. The filters were washedthoroughly with 5% TCA containing 1M Na₂HPO₄/NaH₂PO₄, pH 7.0, rinsedwith water and then ethanol, air dried, and the filter-boundradioactivity was measured in the scintillation counter. By carrying outthe reaction in the presence of various concentrations of each of thecompounds set out above it was possible to determine IC50 values foreach compound with the formula:

% residual activity=100/(1+[I]/IC₅₀)^(s)

where [I] is the inhibitor concentration and “s” is the slope of theinhibition curve.

Test for Inhibition of Hepatitis B Virus Polymerase

Analogous assays employed the polymerase of hepatitis B virus (HBV pol),obtained in the form of viral particles from the sera of HBV positivepatients. These particles contain a polymerase bound to an incompletedouble stranded DNA template. In the assay the incorporation of ³²P-dNTPis measured as radioactivity incorporated in acid insoluble precipitate.

The standard reaction (100 μl) was carried out in a buffer containing 50mM tris/HCl pH 7.5, 30 mM MgCl2 , 1 mM DTT, 100 mM KCl, 0.02% TritonX-100, 1 μCi[³²P] dCTP (300 Ci/mmol, NEN), 1 μM dATP, dTTP, dGTP. After1 hour incubation at 37° C. the reaction was stopped by adding 100 μl of20% TCA and applying samples to DE81 filters. The filters were processedand IC₅₀ values calculated as described above.

Test for Inhibition of Human Immunodeficiency Virus-1 ReverseTranscriptase

Analogous assays employed the reverse transcriptase of HIV (HIV−1RT)from Boehringer Mannhium.

Incorporation of radioactive dTTP was measured as follows. The standardreaction (100 μl) was carried out in a buffer containing 50 mM tris/HClpH 8.2, 2.5 mM MgCl 2, 1 mM DTT, 80 mM KCl, 5 mM EGTA, 0.05% TritonX-100, 1 μCi[3H] dTTP (40 Ci/mmol, NEN), 10 μM UTP and 10 μg/mlpoly(A)/dT (from Pharmacia). The final HIV−1RT (enzyme concentration was1 nM. After 1 hour incubation at 37° C. the reaction was stopped byadding 100 μl of 20% TCA and applying samples to DE81 filters. Thefilters were processed and IC₅₀ values calculated as described above.

The results demonstrate that the compounds of the present invention areeffective as inhibitors of viral polymerases at low micromolarconcentrations.

It is apparent from the tables above that a compound of the presentinvention which is effective in the inhibition of one of the RNAdependent polymerases tested may not necessarily be as effective ininhibiting the other RNA dependent polymerases. The results shown in thetables above indicate a general trend, although this is not withoutexception. Generally, the most active inhibitors of HCV RdRp contained aphenyl ring attached to the diketoacid, whereas the HIV-RT inhibitorscontained a furanyl group and those of HBV polymerase a thiophene group.

While not wishing to be bound by any particular theory, the presentinventors hypothesize that the diketoacid fragment of the compounds ofthe present invention inhibits RNA dependent polymerase activity byproviding an “active site anchor” and interacting with divalent metalcations (Mg²⁺, Mn²⁺) required for polymerase activity. The ring systemfound on the left hand side of the molecule can apparently be modifiedin order to build specificity towards a giver polymerase.

What is claimed is:
 1. A method for for treating or prophylaxis of aviral illness selected from hepatitis C and hepatitis B, which comprisesadministering to a human or animal subject suffering from hepatitis B orhepatitis C a therapeutically or prophylactically effective amount of acompound of Formula A, or a pharmaceutically acceptable salt or esterthereof:

wherein the group R is an organic moiety containing 2 to 24 carbon atomswhich includes an optionally substituted cyclic or heterocyclic group,and wherein one of the atoms in the ring of the cyclic or heterocyclicgroup is directly bonded to the adjacent carbonyl in the diketoacid. 2.The method according to claim 1 wherein in the compound of Formula A thegroup R is selected from: (i) optionally substituted aromatic groups;(ii) optionally substituted heteroaryl groups; (iii) optionallysubstituted cycloalkyl groups; (iv) optionally substituted cycloalkenylgroups; and (v) optionally substituted cyclic heteroalkyl groups.
 3. Themethod according to claim 1, wherein in the compound of Formula A thegroup R is an optionally substituted phenyl group of formula:

wherein R₅ and R₆ independently are selected from hydrogen and thefollowing substituent groups: (a)—OH; (b)—SH; (c)—halogen, (d)—CO₂H;(e)—CN; (f) —NO₂; (g)—NR₁R₂ wherein each of R₁ and R₂ is selected from Hand lower alkyl groups having 1 to 6 carbon atoms; or R₁ and R₂ togetherform a ring including 4 to 6 carbon atoms; (h)—SO₂NR₁R₂ where R₁ and R₂are as defined above; (i)—CONR₁R₂, —NR₁CO₂H, or —NR₁COCOOH where R₁ andR₂ are as defined above; (j) an alkyl (or alkenyl or alkynyl group)group having 1 to 12 (2 to 12) carbon atoms, preferably 1 to 7 (2 to 7)carbon atoms optionally substituted by any one or more of the groups(a)-(i) above and/or optionally interrupted by a group selected from—O—, —S—, —NR₃—, —C(═O)—, —CO₂—, —OCO—, —CONR₃—, —NR₃CONR₃—, —SO₂—,—NR₃SO₂—, and —SO₂NR₃—; where each R₃ independently is H or lower alkylof 1 to 6 carbon atoms; (k) an aryl or heteroaryl group having 2 to 10carbon atoms optionally substituted with any one or more of groups (a)to (j) above; (l) an aralkyl or heteroaralkyl group having 3 to 16carbon atoms optionally substituted with any one or more of groups(a)-(j) above and/or in which the alkyl part of the group is optionallyinterrupted by a group selected from —O—, —S—, —NR₃—, —C(═O)—, —CO₂—,—OCO—, —CONR₃—, —NR₃CONR₃—, —SO₂—, —NR₃SO₂—, and —SO₂NR₃—; where R₃ isas defined above; (m)—C(═O)—R₄ where R₄ is an alkyl, alkenyl, alkynyl,aryl, heteroaryl, aralkyl, or heteroaralkyl group as such groups aredefined above at (j), (k) and (l); (n)—C(═O)—O—R₄ or —O—C(═O)—R₄ whereR₄ is as defined above;. (o)—OR₄ where R₄ is as defined above;(p)—C(═O)NHR₄, —NH—C(═O)—R₄ or —NH—C(═O)—NHR₄ where R₄ is as definedabove; (q)—SO₂R₄ where R₄ is as defined above; (r)—NHR₄ or —N(R₄)₂ whereR₄ is as defined above; (s)—NHSO₂R₄ or —SO₂NHR₄, where R₄ is as definedabove; and (t)—SR₄; and each of optional substituents (j) to (t) abovemay optionally itself be substituted by one or more groups selected from(j) to (t).
 4. The method according to claim 3, wherein the substituentsR₅ and R₆ are independently selected from —H, —OH, —OR₄, —NHSO₂R₄, loweralkyl, aralkyl, amino, amide, urethane groups, and urea groups.
 5. Themethod according to claim 3, wherein the substituents R₅ and R₆ areindependently selected from —H, —OH, —OR₄, and —NHSO₂R₄.
 6. The methodaccording to claim 4, wherein the compound of Formula A contains onlyone substituent either of formula —OR₄ or —NHSO₂R₄.
 7. The methodaccording to claim 4, wherein the compound of Formula A contains a groupof formula —OR₄ and/or —NHSO₂R₄ selected from: —OCH₂Ar; —O(CH₂)₂Ar;—O(CH₂)₃CN; —O(CH₂)₃C═CH; and —NHSO₂Ar; wherein Ar is an optionallysubstituted aryl or heteroaryl group.
 8. The method according to claim3, wherein the compound of Formula A has a single substituent at aposition ortho- or meta- to the diketoacid group.
 9. The methodaccording to claim 3, wherein the compound of Formula A has twosubstituents at the 2,5-; 3,5-; or 2,4-positions.
 10. The methodaccording to claim 2, wherein in the compound of Formula A the group offormula R has the formula:

and each of R₇ and R₈ is independently selected from hydrogen or fromthe list of substituent groups set out at claim 3, and X is O, S, NH orNR₄, where R₄ is as defined above.
 11. The method according to claim 10,wherein the compound of Formula A is a pyrrole-2-substituted diketoacid,a pyrrole-3-substituted diketoacid, a thiophene-2-substituteddiketoacid, or a thiophene-3-substituted diketoacid.
 12. The methodaccording to claim 11, wherein the compound of Formula A is a pyrrolesubstituted diketoacid in which each of R₇ and R₈ is hydrogen.
 13. Themethod according to claim 10, wherein the compound of Formula A is apyrrole substituted dikotoacid having X═NR₄ and wherein R₄ is selectedfrom optionally substituted or interrupted, alkyl aryl or aralkylgroups.
 14. The method according to claim 2, wherein in the compound ofFormula A R is selected from cyclopropyl, cyclopentyl, cyclohexyl,cyclopentenyl, cyclohexenyl and adamantyl groups, any of which may,optionally, be substituted.
 15. The method according to claim 1, whichis a method for treating hepatitis C.
 16. The method according to claim1, which is a method for treating hepatitis B.
 17. The method accordingto claim 1, which is a method for prophylaxis of hepatitis C.
 18. Themethod according to claim 1, which is a method for prophylaxis ofhepatitis B.